WO1992004887A1 - Induction of cytotoxic t cell - Google Patents

Induction of cytotoxic t cell Download PDF

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Publication number
WO1992004887A1
WO1992004887A1 PCT/JP1990/001231 JP9001231W WO9204887A1 WO 1992004887 A1 WO1992004887 A1 WO 1992004887A1 JP 9001231 W JP9001231 W JP 9001231W WO 9204887 A1 WO9204887 A1 WO 9204887A1
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Prior art keywords
antigen
liposome
complex
coated
protein
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PCT/JP1990/001231
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French (fr)
Japanese (ja)
Inventor
Junzo Sunamoto
Hiroshi Shiku
Original Assignee
Kyowa Hakko Kogyo Co., Ltd.
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Priority to PCT/JP1990/001231 priority Critical patent/WO1992004887A1/en
Publication of WO1992004887A1 publication Critical patent/WO1992004887A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a cancer cell antigen or a virus antigen-ribosome complex coated with a polysaccharide containing mannose and a cancer cell or virus antigen using the complex.
  • the present invention relates to a method for inducing a novel cytotoxic T cell (hereinafter abbreviated as “CT”), and a method for treating or preventing cancer or a virus disease using the CTL.
  • CT novel cytotoxic T cell
  • CTL specifically recognizes and impairs cancer cells or virus- and virus-sensitized cells by their antigen receptors, and CTLs are used to detect in vivo cancer or virus. It is also known to be important in host defense mechanisms against Luss.
  • the present invention provides a cancer cell antigen or a virus antigen-liposome complex, which is coated with a polysaccharide containing mannose.
  • antigen refers to substances that elicit an immune response, such as polypeptides, polypeptide complexes, glycoproteins, nucleic acids, and the like. It may be part of the pathological target itself, or may be an antigen expressed by a diseased tissue such as a neoplastic tissue or a virus-infected cell.
  • an antigen protein is preferably used.
  • the antigen protein any origin and purity can be used as long as it includes an antigen determination site, but those prepared by a gene recombination method are preferable.
  • the molecular weight of the antigen protein is not particularly limited as long as it includes at least one antigen-determining site, but is generally 500 to 1,000,000, preferably 200 to 100,000. is there.
  • gag protein and env protein of ATLV include the gag protein and env protein of ATLV, the env protein of HIV, and partial proteins thereof.
  • gag-env fusion protein of ATLV described in the following literature can be mentioned. [JP-A-62-48699; Kuga, T. et al., Japanese 'Journal of Cancer Research., Jpn. J. Cancer Res., 79, 1168-1173 (1988) )].
  • the method of preparing the liposome, the method of encapsulating the antigen in the liposome, and the method of coating the liposome with the Mannos polysaccharide are prepared by the method developed by the present inventors. However, it is also possible to use a general liposome preparation method. [M's Jy's Best Mouth (MJ Ostro), Liposomes, MARCEL DEKER, INC (1987, New York).
  • the mannose-containing polysaccharide may be a chemically synthesized product, a natural extract, or a chemically modified product thereof, as long as mannose is contained as a constituent sugar.
  • readily available mannans and mannan derivatives are used.Particularly, cholesterol residues are partially introduced into mannans for the purpose of producing stable coated polysaccharide ribosomes. It is preferred to do so.
  • JP-A-61-268628 Sunamoto et al., Chem. Lett. 1781 (1988); Takada, A. et al., Biohi Mika. Biochim. Biophys. Acta 802, 237 (1984); Sunamoto, Pathophysiology, 771 (1987)].
  • the liposome can be coated with the mannose-containing polysaccharide by incubating the polysaccharide.ice solution and the liposome at an appropriate temperature.
  • An example of a method for preparing an antigen-liposome complex coated with a mannan derivative is performed by the following procedure. (1) Preparation of liposomes, (2) Modification of liposomes to antigens, (3) Coating of antigen-modified ribosomes with mannan derivatives
  • liposomes formed a bilayer membrane with the same basic structure as cell plasma membrane when Bangham in the United Kingdom redispersed phospholipids derived from natural products in water. Since it was found that it has a Balta structure and closed vesicle morphology, it has been used as a membrane model or as a microcapsule, and it has been reported that the membrane fluidity ⁇ barrier function (membrane permeation barrier) etc. It has been widely used in research on cell structural characteristics and also on research on animal functions of cells such as aggregation and fusion.
  • a liposome having excellent stability and the partial structure of sphingomyelin, which has been developed by the present inventors and is considered to be a kind of boundary lipid in nature, is considered.
  • DDPC j 1.2-dimyristylamide 1.2-dexoxyphosphatidylcholine
  • the above-mentioned liposome is prepared by using various liposome-forming phospholipids and the above-mentioned DDPC, and various methods known per se.
  • lipids for ribosome formation are More specifically, for example, lipids having an amide group capable of forming a hydrogen bond zone in a membrane of lecithin, sphingomyelin, etc. extracted and purified from soybean, egg yolk, etc. And preferably yolk lecithin, dimyristylphosphatidylzircholine (DMPC), etc.
  • a particularly preferred liposome is a combination of yolk lecithin and DDPC It is usually prepared by combining DMPC and DDPC in a ratio of about 4: 6 or 2: 8 (weight ratio).
  • the modification of the antigen to the ribosome is basically performed by simply mixing the two, that is, adding a predetermined amount of the antigen suspension to the ribosome suspension. It can be performed by mixing.
  • the amount of antigen added to the liposome in the above is very important in relation to the ability to express the activity of the obtained complex, and is generally from 10 to L000, preferably about 200 to 10 mg of lecithin in 1 Use the antigen mass.
  • the coating with a mannan derivative is basically performed by simply mixing the two, that is, adding a predetermined amount of a cholesteryl-modified mannan permanent solution to the antigen-modified liposome suspension and mixing.
  • the mass of mannan derivative is 0.1 to 10 mg, preferably about 2 mg per 10 mg of lecithin in 1 mi.
  • antigen-ribosome complex coated with mannose monosaccharide polysaccharide can be used in the present invention as it is, but of course-if necessary, sterilization or the like may be carried out according to a conventional method. Operations can also be performed.
  • the liposome complex of the present invention can be immunized by administering a predetermined value to the animal in the same manner as other conventionally known pectins.
  • the animal to which the vaccine can be applied is not particularly limited, and may be any of various mammals including humans and other animals. Immunization of these animals by administration of the vaccine of the present invention can also be performed according to a general method.
  • the route of administration of the vaccine of the present invention is generally subcutaneous injection, but is not particularly limited thereto.
  • the dose of the pectin of the present invention for the above immunization can be determined as appropriate, but usually the dose is about 50 to: L 50 #g Z ratton times as the antigen. It is appropriate to administer about 1 to 4 times.
  • FIG. 1 shows the results of a comparison of the effect of a mannan-coated antigen-liposome complex on TARS-1 cells.
  • A is a physiological saline
  • B is a mannan-coated antigen-ribosome complex (the present invention)
  • C is a non-mannan-coated antigen-liposome complex
  • D is And the results obtained when the liposomes coated with mannan were respectively administered.
  • FIG. 2 shows a comparison result of induction of CTL activity of the antigen-liposome complex coated with mannan.
  • A is a physiological saline
  • B is a mannan-coated antigen-ribosome complex (the present invention)
  • C is an unmannan-coated antigen-ribosome complex
  • D is a man-ribosome complex.
  • EggPC Egg yolk phosphatid zirconine
  • gag-env fusion protein aqueous solution 1 rafi 200 as protein
  • the gag-env fusion protein aqueous solution 1 rafi 200 as protein
  • the mixture was mixed and incubated at 371: 6 for 6 hours to obtain an antigen-encapsulated liposome suspension 2.
  • physiological saline A in FIG. 1
  • C in FIG. 1 A group was also administered with mannan-coated ribosome alone (D in FIG. 1).
  • Rats were immunized as in Example 2. 1 week after the last dose Later, spleen cells of the test animals were collected. 4 ⁇ 10 7 spleen cells and mitomycin in RPM1 1640 medium 20 ⁇ containing 20% fetal calf serum. 4 x 10 s of the treated TARS-1 cells were co-cultured at 37: for 1 week in a carbon dioxide incubator. The cytotoxicity after culturing was determined by the 51 Cr release method [Nakayama, E. et al., Proc. Of the National University of California, California, Science, USA (Proc. Natl. Acad. Sci. USA) 76, 3486 (1979)] (Fig. 2, B).
  • physiological saline A in FIG. 2
  • an antigen-ribosomal complex not coated with a mannan derivative FOG. 2
  • C ribosome alone coated with a mannan derivative

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Abstract

A complex of a cancer cell antigen or a viral antigen with liposome covered with a polysaccharide including mannose, and a method of inducing a cytotoxic T cell specific for a cancer cell or a virus antigen by using said complex.

Description

明 細 書  Specification
細胞障害性 τ細胞の誘導方法  Methods for inducing cytotoxic τ cells
技 術 分 野  Technical field
本発明は、 マンノ ースを舍む多糖類で被覆された、 がん細胞抗原ま たはウィ ルス抗原一リ ボソーム複合体ならびに該複合体を用いる該が ん細胞またはウ ィ ルス抗原に特異的な細胞障害性 T細胞 （以下 "CT と略記する） の誘導方法ならびに、 その C T Lによるがんまたはウイ ルス疾患の治療または予防方法に関する。  The present invention relates to a cancer cell antigen or a virus antigen-ribosome complex coated with a polysaccharide containing mannose and a cancer cell or virus antigen using the complex. The present invention relates to a method for inducing a novel cytotoxic T cell (hereinafter abbreviated as “CT”), and a method for treating or preventing cancer or a virus disease using the CTL.
背 景 技 術  Background technology
C T Lはがん細胞も しく はウイ ルスならびにゥィ ルス感作細胞を、 その抗原リ セプターにより特異的に認識し障害することは知られてお り、 C T Lは生体内でのがんも しく はウ ィ ルスに対する生体防御機構 において重要であることも知られている。  It is known that CTL specifically recognizes and impairs cancer cells or virus- and virus-sensitized cells by their antigen receptors, and CTLs are used to detect in vivo cancer or virus. It is also known to be important in host defense mechanisms against Luss.
〔ディ レイ ' ェ厶 ' ォ一 （ Da i I ey， M.0. )ら、 プロ シーデイ ング ' ォブ ' ナショ ナル ♦ アカデミ ー ' ォブ ' サイ エンス ' USA (Pro Natl. Acad. Sci. USA) 79 , 5384—5387 (1984)； ナカヤマ ' ィ ー（Nakayama, Ε. ) ら、 ジャ ーナル ' ォブ ' ェクスペ リ メ ンタル ， メディ ス ン（J. Exp. Med, ) 161 , 345-355 (1985)； グ リ一ンバーグ · ピー ' ォー（Greenberg, P.0. )、 ジャ ーナル . ォブ · ィ ミ ュ ノ ロ ジー（J. Immunol. ) 136 , 1917-1922 (1986) J ； ク ラ 一ネ ッ ト ' ジヱイ ' ピー （Klarnet, J. Ρ· )ら、 ジャ ー ナル ' ォブ ' イ ミ ュノ ロ ジ一 U. Immunol. ) 138, 4012-4017 (1987)； フ ォ ン ' デル ' ホーン（Von der Hoorn) ら、 ジャ ーナル · ォブ ' ェク スペ リ メ ンタル ' メディ スン（J. Exp. Med. ) 162, 123-144 (1985) 〕 c さ らに、 最近の研究によって、 ガン細胞も しく はウィ ルスならびに ゥィ ルス感作細胞の抗原性タ ンパク質は、 単独で提示されても C T L は誘導されず、 抗原提示細胞上に MH Cク ラス I と共発現された場合 のみ C T L誘導が可能となることが明らかとなった 〔モ リ ソ ン ' エル ' エー （Morrison, し丄，） ら、 ジャ ーナル · ォ ブ ' ェク スペ リ メ ンタ ル . メ ディ ス ン （J. Bxpjed. ), 163 , 903-921 (1986) ； ガーメ イ ン ' アール . ェヌ （Germain, RJ. )、 ネイチヤ ー （Nature), 322 , 687-689 (1986)] 。 [Dai Iey, M.0., Et al., Proceeding 'ob' National ♦ Academic 'ob' Science 'USA (Pro Natl. Acad. Sci. USA) 79, 5384-5387 (1984); Nakayama, Ε., Et al., Journal of ォ ob 'experimental, Medicine (J. Exp. Med,) 161, 345-355. (1985); Greenberg, P.0., J. Immunol. 136, 1917-1922 (1986) J; Klarnet, J. Ρ, et al., Journal 'ob' Immunology U. Immunol.) 138, 4012-4017 (1987); Von der Hoorn et al., Journal of ブ Bec-Experimental 'Medicine (J. Exp. Med.) 162, 123-144 (1985)) c Furthermore, recent studies have shown that the antigenic protein of cancer cells or virus and virus-sensitized cells does not induce CTLs when presented alone, and that MHC It was found that CTL induction was possible only when co-expressed with class I [Morrison, et al., Et al. J. Bxpjed., 163, 903-921 (1986); Garmain, RJ., Nature, 322, 687-689. (1986)].
従って、 これまでは、 C T L誘導のためには、 ガン細胞そのもの、 もしく はウ ィ ルスまたはウイ ルス感作細胞そのものを用いる必要があ り、 それらの入手の函難さや感染性のため、 応用が制限されていた。 一方、 遺伝子組換え技術の発展により、 がん特異抗原やウ ィ ルス特 異抗原の安全かつ大量生産は可能であるが、 遺伝子組換え技術により 得られた抗原ポ リ べプチ ドを用'いた効率的な CTし 誘導方法はいまだ見 出されていない。  Therefore, to date, it has been necessary to use cancer cells themselves, or virus or virus-sensitized cells themselves, for CTL induction. Was restricted. On the other hand, with the development of genetic recombination technology, it is possible to produce cancer-specific antigens and virus-specific antigens safely and in large quantities, but they used antigen polypeptides obtained by genetic recombination technology. No efficient CT guidance method has been found yet.
発 明 の 開 示 Disclosure of the invention
本発明は、 マ ンノ ースを含む多糖類で被覆された、 がん細胞抗原ま たはウ イ ルス抗原ーリ ポソ 一厶複合体を提供する。  The present invention provides a cancer cell antigen or a virus antigen-liposome complex, which is coated with a polysaccharide containing mannose.
"抗原" という語句はポ リぺプチ ド、 ポ リぺプチ ド複合体、 グリ コ プロティ ン、 核酸等、 免疫応答を惹起する物質を指す。 病理学的標的 そのものの一部であってもよく、 または新生物組織またはウ ィ ルス感 染細胞等の病んだ'組織が発現する抗原であってもよい。 本発明におけ る抗原と しては、 抗原タ ンパク質が好適に用いられる。 抗原タ ンパク質は、 抗原決定部位を含めば、 いかなる起原、 純度の ものでも用いることが可能であるが、 望ま し く は遺伝子組換え法によ り作成したものが好適である。 抗原タ ンパク質の分子量は、 抗原決定 部位を少な く と も一ヶ所以上含めば、 特に限定しないが、 一般的に 500 〜 1, 000, 000 、 好ま し く は 2, 000〜100, 000 である。 The phrase "antigen" refers to substances that elicit an immune response, such as polypeptides, polypeptide complexes, glycoproteins, nucleic acids, and the like. It may be part of the pathological target itself, or may be an antigen expressed by a diseased tissue such as a neoplastic tissue or a virus-infected cell. As the antigen in the present invention, an antigen protein is preferably used. As the antigen protein, any origin and purity can be used as long as it includes an antigen determination site, but those prepared by a gene recombination method are preferable. The molecular weight of the antigen protein is not particularly limited as long as it includes at least one antigen-determining site, but is generally 500 to 1,000,000, preferably 200 to 100,000. is there.
具体例と しては、 ATLVの gag タ ンパク質、 env タ ンパク質、 HIV の env タ ンパク質ならびにそれらの部分タ ンパク質などがあげられる。 Specific examples include the gag protein and env protein of ATLV, the env protein of HIV, and partial proteins thereof.
〔ジヱイ · シィ プノ、'ッノヽ U. Schupbach) 編、 カ レ ン ト · ト ピッ ク ス ' イ ン ' マイ ク ロ ノ ィ ォロ ジ一 ' アン ド ♦ イ ミ ュ ノ ロ ジ一 （ Current Topics in Microbiology and Immunology) , 142 (1989) , スプ リ ンガ 一一バーグ （Springer-Verlag) ；ピー ' ケー ' フォ ー （P. Vogt)編、 カ レン ト · ト ピッ クス ' イ ン . マイ ク ロノ ィ ォロ ジ一 ' アン ド ' ィ ミ ュ ノ 。 ジー (Current Topics in Microbiology and Immunology) , 115 (1985), スプ リ ンガ——バーグ（Springer- Verlag) 〕 c [Gipy Sipno, 'Knod U. Schupbach', ed., Current Topics 'In', My Chromology 'And' ♦ Immunology Topics in Microbiology and Immunology), 142 (1989), Springer-Verlag; Ed. P. Vogt, Current Topics in Mic. Ronoolo's 'and' imiuno. G (Current Topics in Microbiology and Immunology), 115 (1985), Springer-Verlag) c
さ らに、 複数の抗原決定部位を持つ例と しては以下の文献記載の ATLVの gag- env 融合タ ンパク質をあげることができる。 〔特開昭 62 - 48699 ； クガ ♦ ティ 一（Kuga， T. ) ら、 ジャパニーズ ' ジャ ーナル · ォ ブ . キャ ンサー . リ サーチ （Jpn. J. Cancer Res. ), 79 , 1168-1173 (1988)] 。  Further, as an example having a plurality of antigen-determining sites, a gag-env fusion protein of ATLV described in the following literature can be mentioned. [JP-A-62-48699; Kuga, T. et al., Japanese 'Journal of Cancer Research., Jpn. J. Cancer Res., 79, 1168-1173 (1988) )].
リ ポソ一厶の作成ならびに、 抗原の リ ポソームへの封入方法ならび に、 該 リ ボソ ームのマンノ一ス舍有多糖類による被覆方法は、 本発明 者らの開発した方法により作成することが好ま しいが、 一般的な リ ポ ソ 一ム調製法を用いても可能である。 〔ェ ム ' ジヱ イ · ォス ト 口 （M. J. Ostro)編、 リ ポソ一厶ズ（Liposomes), マーセル ' デッカー（MARCEL DEK ER), INC(1987, New York 〕 。 The method of preparing the liposome, the method of encapsulating the antigen in the liposome, and the method of coating the liposome with the Mannos polysaccharide are prepared by the method developed by the present inventors. However, it is also possible to use a general liposome preparation method. [M's Jy's Best Mouth (MJ Ostro), Liposomes, MARCEL DEKER, INC (1987, New York).
マ ンノ ース含有多糖類は、 構成糖と してマ ンノ ースを舍めば、 化学 合成品、 天然抽出物いずれでもよいし、 さ らにそれらの化学修飾物で もよい。 好ま しく は、 入手の容易なマ ンナ ンならびにマ ンナ ン誘導体 が用いられ、 特に、 安定な被覆多糖類リ ボソ ームとする目的で、 マ ン ナン中に一部、 コ レステロール残基を導入することが好適である。 〔特開昭 61- 268628; 砂本ら、 ケ ミ ス ト リ ー · レタ ー （Chem. Lett. ) 1781 (1988) ； タカダ ' ェ厶 （Takada, ϋ ) ら、 バイ オヒ ミカ . バイ オ フイ ジ力 ' ァクタ （Biochim. Biophys. Acta) 802 , 237 (1984) ；砂本、 病態生理 , 771 (1987)] 。  The mannose-containing polysaccharide may be a chemically synthesized product, a natural extract, or a chemically modified product thereof, as long as mannose is contained as a constituent sugar. Preferably, readily available mannans and mannan derivatives are used.Particularly, cholesterol residues are partially introduced into mannans for the purpose of producing stable coated polysaccharide ribosomes. It is preferred to do so. [JP-A-61-268628; Sunamoto et al., Chem. Lett. 1781 (1988); Takada, A. et al., Biohi Mika. Biochim. Biophys. Acta 802, 237 (1984); Sunamoto, Pathophysiology, 771 (1987)].
マ ンノ ース含有多糖類による該リ ポソ ームの被覆方法は、 多糖類.氷 溶液と該リ ポソームを適当な温度でィ ンキュベー トすれば良い。  The liposome can be coated with the mannose-containing polysaccharide by incubating the polysaccharide.ice solution and the liposome at an appropriate temperature.
マ ンナ ン誘導体により被覆さ'れた抗原ーリ ポソ ーム複合体の作成法 の一例は以下の手順で行う。 すなわち、 ①リ ポソームの作成、 ②リ ポ ソ 一ムへの抗原の修飾、 ③抗原修飾リ ボソ ームのマ ンナン誘導体によ る被覆  An example of a method for preparing an antigen-liposome complex coated with a mannan derivative is performed by the following procedure. (1) Preparation of liposomes, (2) Modification of liposomes to antigens, (3) Coating of antigen-modified ribosomes with mannan derivatives
①リ ポソ ームの作成  ① Creation of reposome
リ ボソームは、 1960年中期に英国のバンガム （Bangham) が天然物 由来のリ ン脂質を水中に再分散させたとき、 細胞の形質膜と同じ基 本構造の二分子膜が形成され、 これが細胞のバルタ構造と閉鎖型小 胞形態を有することを見出して以来、 一膜モデルとして乃至はマイ ク ロカプセルとして、 膜の流動性ゃバリャ一能 （膜透過障壁） 等の 細胞構造特性の研究材料や、 また凝集、 融合等の細胞の動物機能研 究等に広く利用され盛んな研究が展開されてきている。 In the mid-1960s, liposomes formed a bilayer membrane with the same basic structure as cell plasma membrane when Bangham in the United Kingdom redispersed phospholipids derived from natural products in water. Since it was found that it has a Balta structure and closed vesicle morphology, it has been used as a membrane model or as a microcapsule, and it has been reported that the membrane fluidity ゃ barrier function (membrane permeation barrier) etc. It has been widely used in research on cell structural characteristics and also on research on animal functions of cells such as aggregation and fusion.
本発明の実施にあたっては、 安定性の優れた リ ポソー厶を用いる ことが好ま しく 、 本発明者らの開発した天然における境界脂質の一 種と考えられているス フィ ンゴミ エ リ ンの部分構造アナログと して、 了 シル類中にア ミ ド結合を有する 1. 2 —ジ ミ リ ス ト ィ ルア ミ ドー 1. 2 —デォキシホスフ ァチジルコ リ ン （以下 「 D D P C j という） を 用いることが好適である （特開昭 61-267509 号公報） 。 より詳しく は上記リ ポソ ー厶は、 通常のリ ポソ 一厶形成様リ ン脂質と上記 DDPC とを利用して、 それ自体公知の各種方法、 例えば代表的にはボルテ ッ クス ( V 0 r t e X 1 n g method又は Hydrat ι on method) (Bangham, A. D. , Standish, M. M. & Watkins, J. C. J. Mol. Biol. , 13 , 238 (1965) 〕 に 従って実施できる。 上記リ ボソ ーム形成用脂質と しては、 より具体 的には例えば、 大豆、 卵黄等'から抽出、 精製された レ シチ ン、 ス フ ィ ゴミ エ リ ン等の膜中で水素結合帯形成を行い得るア ミ ド基を有す る脂質、 好ま し く は卵黄レ シチ ン、 ジ ミ リ ス ト イ ルホス フ ァ チ ジル コ リ ン （ D M P C )等を例示できる。 上記において、 特に好ま しい リ ポソー厶は、 卵黄レシチンと D D P Cとを併用するか又は DM P C と D D P Cとを通常約 4 ： 6または 2 ： 8 (重量比） で併用して調 製される。  In carrying out the present invention, it is preferable to use a liposome having excellent stability, and the partial structure of sphingomyelin, which has been developed by the present inventors and is considered to be a kind of boundary lipid in nature, is considered. As an analog, it is preferable to use 1.2-dimyristylamide 1.2-dexoxyphosphatidylcholine (hereinafter referred to as “DDPC j”) having an amide bond in a polysyl group. More specifically, the above-mentioned liposome is prepared by using various liposome-forming phospholipids and the above-mentioned DDPC, and various methods known per se. For example, typically performed according to Vortex (V 0 rte X 1 ng method or Hydrat ion method) (Bangham, AD, Standish, MM & Watkins, JCJ Mol. Biol., 13, 238 (1965)) The above lipids for ribosome formation are More specifically, for example, lipids having an amide group capable of forming a hydrogen bond zone in a membrane of lecithin, sphingomyelin, etc. extracted and purified from soybean, egg yolk, etc. And preferably yolk lecithin, dimyristylphosphatidylzircholine (DMPC), etc. In the above, a particularly preferred liposome is a combination of yolk lecithin and DDPC It is usually prepared by combining DMPC and DDPC in a ratio of about 4: 6 or 2: 8 (weight ratio).
リ ポソ ームへの抗原の修飾 Modification of antigen to liposome
リ ボソ ームへの抗原の修飾は、 基本的には、 両者を単に混合する ことにより、 即ち、 リ ボソ ーム懸濁液に所定値の抗原懸濁液を添加 混合することにより実施できる。 The modification of the antigen to the ribosome is basically performed by simply mixing the two, that is, adding a predetermined amount of the antigen suspension to the ribosome suspension. It can be performed by mixing.
上記における リ ポソームに対する抗原の添加量は得られる複合体 の活性発現能と関連して非常に重要であり、 一般には 1 中のレシ チン 10mgに対して 10〜： L 000 、 好ましく は約 200 の抗原質量とす る。  The amount of antigen added to the liposome in the above is very important in relation to the ability to express the activity of the obtained complex, and is generally from 10 to L000, preferably about 200 to 10 mg of lecithin in 1 Use the antigen mass.
③抗原修飾リ ボソ ームのマ ンナン誘導体による被覆  ③ Coating of antigen-modified ribosome with mannan derivative
マ ンナン誘導体による被覆は、 基本的には、 両者を単に混合する ことにより、 即ち、 抗原修飾リ ポソ ーム懸濁液に所定量のコ レステ πール修飾マンナン永溶液を添加、 混合することにより実施できる < 一般的には 1 mi?中のレシチン 10mgに対して 0. l〜10mg、 好ま しく約 2 mgのマ ンナ ン誘導体質量とする。  The coating with a mannan derivative is basically performed by simply mixing the two, that is, adding a predetermined amount of a cholesteryl-modified mannan permanent solution to the antigen-modified liposome suspension and mixing. <Generally, the mass of mannan derivative is 0.1 to 10 mg, preferably about 2 mg per 10 mg of lecithin in 1 mi.
このようにして得られたマ ンノ 一ス舍有多糖類で被覆された抗原 一リ ボソ ーム複合体は、 そのまま本発明に利用できるが、 もちろん- 必要に応じて、 常法に従い滅菌等の操作を施すこともできる。  The thus obtained antigen-ribosome complex coated with mannose monosaccharide polysaccharide can be used in the present invention as it is, but of course-if necessary, sterilization or the like may be carried out according to a conventional method. Operations can also be performed.
本発明の該リ ポソーム複合体は従来公知の他のヮクチンと同様に して、 その所定値を動物に投与することにより、 該動物を免疫化す ることができる。 該ワ ク チ ンを適用される得る動物は特に限定はな く、 ヒ トを初めとする各種の哺乳動物、 その他の動物のいずれでも よい。 またこれら動物の本発明ワクチン投与による免疫化も、 一般 的方法に従う ことができる。 本発明ワクチンの投与経路としては、 皮下注射が一般的であるが、 特にこれに限定されるものではない。 上記免疫化に当たつての本発明ヮクチンの投与量は、 適宜決定でき るが、 通常投与量は、 抗原として 50〜： L 50#g Zラ ッ トノ回程度の量 を目安とし、 投与は 1 〜 4回程度行うのが適当である。 The liposome complex of the present invention can be immunized by administering a predetermined value to the animal in the same manner as other conventionally known pectins. The animal to which the vaccine can be applied is not particularly limited, and may be any of various mammals including humans and other animals. Immunization of these animals by administration of the vaccine of the present invention can also be performed according to a general method. The route of administration of the vaccine of the present invention is generally subcutaneous injection, but is not particularly limited thereto. The dose of the pectin of the present invention for the above immunization can be determined as appropriate, but usually the dose is about 50 to: L 50 #g Z ratton times as the antigen. It is appropriate to administer about 1 to 4 times.
図面の簡単な説明  BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 マ ンナ ン被覆した抗原ーリ ポソ ーム複合体の T A R S— 1細胞に対する効果を比較した結果を示す。  FIG. 1 shows the results of a comparison of the effect of a mannan-coated antigen-liposome complex on TARS-1 cells.
Aは、 生理食塩水、 Bは、 マ ンナ ン被覆した抗原一リ ボ ソ ーム複合 体 （本発明） 、 Cは、 マ ンナン被覆されていない抗原ーリ ポソ 一ム複 合体、 Dは、 マ ンナ ン被覆した リ ポソー厶をそれぞれ投与した場合の 結果である。  A is a physiological saline, B is a mannan-coated antigen-ribosome complex (the present invention), C is a non-mannan-coated antigen-liposome complex, D is And the results obtained when the liposomes coated with mannan were respectively administered.
第 2図は、 マンナン被覆した抗原ーリ ポソーム複合体の C T L活性 誘導の比較結果を示す。  FIG. 2 shows a comparison result of induction of CTL activity of the antigen-liposome complex coated with mannan.
Aは、 生理食塩水、 Bは、 マ ンナン被覆した抗原一リ ボ ソ ーム複合 体 （本発明） 、 Cは、 マ ンナン被覆されていない抗原一リ ボソ ーム複 合体、 Dは、 マ ンナ ン被覆したリ ボソ ームをそれぞれ投与した場合の 結果である。 '  A is a physiological saline, B is a mannan-coated antigen-ribosome complex (the present invention), C is an unmannan-coated antigen-ribosome complex, and D is a man-ribosome complex. The results were obtained when each of the ribosomes coated with nannan was administered. '
癸明を実施するための最良の形態 The best form for carrying out KIKI
以下、 本発明を更に詳しく説明するための実施例をあげる。  Hereinafter, examples for describing the present invention in more detail will be described.
実施例 1 マ ンナ ン誘導体で被覆された抗原ーリ ポソ ーム複合体の調 製 Example 1 Preparation of antigen-liposome complex coated with mannan derivative
1 ) リ ポソ ー厶の調製  1) Preparation of liposome
卵黄から文献記載の方法 (W. S. Singleton, M. S. Gray, Μ·し. Brown, J. L. White, J. Am. Oil Chem. Soc, , 42 ， 53 (1965) 〕 に従い卵黄ホス フ ァ チ ジルコ リ ン （EggPC) を抽出し、 アル ミ ナカ ラ ムを用いて精製 した。 その純度は T L Cにより確認した。 また、 2, 3 —ジァ ミ ノ プロ ピオ ン酸から特開昭 61-267509号公報 (砂本順三ら、 日本化学会誌、 1987 (3) 、 P569-574) 記載の方法に より、 D D P Cを合成した。 Egg yolk phosphatid zirconine (EggPC) according to the method described in the literature (WS Singleton, MS Gray, Μ · Sh. Brown, JL White, J. Am. Oil Chem. Soc,, 42, 53 (1965)) ) Was extracted and purified using alumina.The purity was confirmed by TLC. Further, DDPC was obtained from 2,3-diaminopropionic acid by the method described in JP-A-61-267509 (Junzo Sunamoto et al., Journal of the Chemical Society of Japan, 1987 (3), P569-574). Was synthesized.
上記 EggPC の 10.6mgと D D P Cの 7.0mgとを混合し、 ナス型フラ ス コ中で 2 mgのク ロ口ホル厶中に溶解させた。 ロータ リ ーエバボレ 一ターを用いてクロ 口ホルムを減圧下に留去し、 更に減圧デシケー タ—中で—夜放置した。  10.6 mg of the above-mentioned EggPC and 7.0 mg of DDPC were mixed, and dissolved in 2 mg of clog-mouthed form in an eggplant-shaped flask. The crotch form was distilled off under reduced pressure using a rotary evaporator, and the mixture was allowed to stand overnight in a reduced pressure desiccator.
次いで得られた溶液をボルテツ ク ス ミキサー上で緩衝液 A (lOmM 炭酸緩衝液、 0.35 尿素、 0.07mM EDTA 、 0.035¾ 2-メ ルカプトェ タ ノ ール、 ΡΗ?. 4)の 1 に膨潤させ、 剝雜した。 その後、 UR200Pプ 口ーブ型超音波破枠機 （ト ミ —社製） を用いて、 窒素ガス気流下に 5分間、 0 で、 25Κ で超音波照射して、 所望のリ ポソ ーム懸濁液を ) リ ボソ ームへの抗原の修 r  The resulting solution was then swollen on a vortex mixer into 1 of buffer A (10 mM carbonate buffer, 0.35 urea, 0.07 mM EDTA, 0.035¾2-mercaptoethanol, ΡΗ? .4), I was crowded. Then, using a UR200P probe ultrasonic breaker (manufactured by Tomi Corporation), irradiate ultrasonic waves at 25 ° at 0 ° for 5 minutes under a nitrogen gas flow to obtain the desired liposome Suspension) Repair of antigen to ribosome
特開昭 62- 48699号公報記載の方法により作成した の gag-env 融合タ ンパク質水溶液 1 rafi (タ ンパク質として 200 ） と上記 1 ) で調製した リ ポソ一ム懸濁液 1 m2とを混合し、 371：で、 6時間イ ン キュベ一 ト して、 抗原封入リ ポソ 一ム懸濁液 2 を得た。  The gag-env fusion protein aqueous solution 1 rafi (200 as protein) prepared by the method described in JP-A-62-48699 and the liposome suspension 1 m2 prepared in 1) above were used. The mixture was mixed and incubated at 371: 6 for 6 hours to obtain an antigen-encapsulated liposome suspension 2.
) マ ンナ ン誘導体で被覆された抗原ーリ ポソ ーム複合体の調製 上記 '2 ) で得られた リ ポソ一ム懸濁液 2 m2に、 下記の文献記載の 方法により作成したコ レステロール修飾マ ンナンの 5 mgZra2水溶液 0.5 mgを加え、 室温で 30分間ィ ンキュベー ト し、 目的物を得た 〔T, Sato, . o j ima, T. I hda, and J. Sunatnoto, Macrophage Activation by Poly (maleicacid-alt-z-c clohe y 1-1, 3-d i oxap-5- ene) Encapsulated in Pol saccharide- Coated Liposomes, J. ) Preparation of antigen-liposome complex coated with mannan derivative Two ml of the liposome suspension obtained in '2) above was prepared by the method described in the following literature. 0.5 mg of a 5 mg aqueous solution of resterol-modified mannan was added, and the mixture was incubated at room temperature for 30 minutes to obtain the desired product (T, Sato, .ojima, T.I hda, and J. Sunatnoto, Macrophage Activation by Poly (maleicacid-alt-zc clohe y 1-1, 3-di oxap-5-ene) Encapsulated in Pol saccharide- Coated Liposomes, J.
Bioactive and Compatible Polym. , _1, 448-460 (1986)：· c Bioactive and Compatible Polym., _1, 448-460 (1986): c
実施例 2 マ ンナ ン誘導体で被覆された抗原ーリ ポ リ ーム複合体の効 果 I 〔イ ン . ビボ （ i_vo )効果〕  Example 2 Effect of Antigen-Liposome Complex Coated with Mannan Derivative I [In Vivo (i_vo) Effect]
W A/H 雌性ラ ッ ト 1群 4匹に、 実施例 1 で調製した リ ポソーム複 合体懸濁液 lOOmgを、 1週間間隔で 2 回皮下に投与して C T Lを誘導 させ、 最終投与から 1週間後に、 供試動物の皮内に、 文献 〔ノ グチ - ヮ ィ （Noguchi, Ϊ. )ら、 ジャ ーナリレ ' ォブ . イ ミ ュ ノ ロ ジー （J. Immunol. ) 143 , 3737-3742 (1989) 〕 記載の方法により調製した T A R S— 1細 胞 1 X 107個を移植した。 WA / H female rats per group, 4 animals, were subcutaneously administered twice a week with the liposome complex suspension lOOmg prepared in Example 1 at 1-week intervals to induce CTL, and 1 week after the last administration Later, in the skin of the test animal, the literature [Noguchi, Ϊ., Et al., Jannarele ob., I. Immunol., 143, 3737-3742 (1989) 1) 1 × 10 7 TARS-1 cells prepared by the method described above were transplanted.
その後、 移植した T A R S— 1細胞のコロ ニ一の直径を経曰的に測 定して、 効果を判定した。 （第 1 図の B)  Thereafter, the diameter of the colony of the transplanted TAR S-1 cells was measured by the method described above to determine the effect. (B in Fig. 1)
比較及び対照と して、 該リ ボソ ーム複合体に代えて、 生理食塩氷 (第 1図の A) 、 マ ンナ ン非被覆の抗原一リ ボソ ーム複合体 （第 1 図 の C ) および、 マ ンナ ン被覆した リ ボ ソ ーム単独 （第 1図の D) をそ れぞれ同様に投与した群を設けた。  As a comparison and a control, instead of the ribosome complex, physiological saline (A in FIG. 1) and a non-mannan-coated antigen-ribosome complex (C in FIG. 1) were used. A group was also administered with mannan-coated ribosome alone (D in FIG. 1).
これらの結果、 本発明の抗原ーリ ポソ ーム複合体投与群においては、 腫瘍の増殖はほとんど認められず、 顕著な腫瘍増殖抑制効果を奏する ことが判明した。  As a result, in the group administered with the antigen-liposome complex of the present invention, almost no tumor growth was observed, and it was found that a significant tumor growth inhibitory effect was exhibited.
実施例 3 マ ンナン誘導体で被覆された抗原一リ ボ ソ ーム複合体の効 果 Π 〔イ ン · ビ ト ロ （in vitro ) 効果〕 Example 3 Effect of antigen-ribosome complex coated with mannan derivative [In vitro effect]
実施例 2 と同様に、 ラ ッ トの免疫化を行った。 最終投与から 1週間 後に、 供試動物の脾臓細胞を集めた。 20% 牛胎児血清を含む RPM1 1640 培地 20 ^中で、 上記脾臓細胞 4 X 107個とマイ トマイ シ ン。処理した T A R S— 1細胞 4 X 10 s個を、 炭酸ガスイ ンキュベーターを用い、 37 :で 1週間、 共培養した。 培養後の細胞障害性を51 Cr放出法 〔ナカ ャマ ' ィ 一 （Nakayama, E) ら、 プロ シーデイ ング . ォブ ' ナショナル • ァ力デミ ィ 一 * ォブ ' サイ エ ンス ' USA (Proc. Natl. Acad. Sci. USA) 76 , 3486 (1979) 〕 により分析した （第 2図の B) 。 比較及び対照 と して、 該リ ポソ 一ム複合体に代えて、 生理食塩水 （第 2図の A ) 、 マ ンナ ン誘導体非被覆の抗原一リ ボソ ーム複合体 （第 2図の C) およ び、 マ ンナン誘導体で被覆したリ ボソ ーム単独 （第 2図の D) をそれ ぞれ同様に投与した群を設けた。 Rats were immunized as in Example 2. 1 week after the last dose Later, spleen cells of the test animals were collected. 4 × 10 7 spleen cells and mitomycin in RPM1 1640 medium 20 ^ containing 20% fetal calf serum. 4 x 10 s of the treated TARS-1 cells were co-cultured at 37: for 1 week in a carbon dioxide incubator. The cytotoxicity after culturing was determined by the 51 Cr release method [Nakayama, E. et al., Proc. Of the National University of California, California, Science, USA (Proc. Natl. Acad. Sci. USA) 76, 3486 (1979)] (Fig. 2, B). As a comparison and a control, instead of the liposomal complex, physiological saline (A in FIG. 2) and an antigen-ribosomal complex not coated with a mannan derivative (FIG. 2) were used. C) and ribosome alone coated with a mannan derivative (D in FIG. 2) were similarly administered.
第 2図において明らかなように、 本発明のマ ンナ ン誘導体で被覆し た抗原一リ ボソ ーム複合体の場合 （第 2図の B) のみ、 高い C T L活 性が誘導された。  As is clear from FIG. 2, high CTL activity was induced only in the case of the antigen-ribosome complex coated with the mannan derivative of the present invention (FIG. 2B).

Claims

請 求 の 範 囲 The scope of the claims
1. マ ンノ ースを舍む多糖類で被覆された、 がん細胞抗原またはウイ ルス抗原ーリ ポソ ーム複合体。  1. A cancer cell antigen or virus antigen-liposome complex coated with a polysaccharide that contains mannose.
2. 該抗原が成人 T細胞白血病ウ ィ ルス （以下 "A T L V " と略記す る） の gagタ ンパク質、 env タ ンパク質またはそれらの一部である 請求項 1記載の複合体。  2. The complex according to claim 1, wherein the antigen is gag protein, env protein, or a part thereof of adult T-cell leukemia virus (hereinafter abbreviated as "ATLV").
3. 該抗原が A T L Vの gag- env融合タ ンパク質である請求項 1記載 の複合体。  3. The complex according to claim 1, wherein the antigen is an ATLV gag-env fusion protein.
4. マ ンノ ースを含む多糖類で被覆された、 がん細胞またはウ ィ ルス 抗原ーリ ポソ 一ム複合体をヒ トまたは動物に投与し、 ヒ トまたは動 物中に該がん細胞抗原またはウ ィ ルス抗原に特異的な細胞障害性 T 細胞を誘導する方法。  4. Administer a cancer cell or virus antigen-liposome complex coated with a polysaccharide containing mannose to a human or animal, and obtain the cancer in the human or animal. A method for inducing cytotoxic T cells specific for cellular or viral antigens.
5. 該抗原が A T L Vの gagタ ンパク質、 envタ ンパク質またはそれ らの一部である請求項 4記載の方法。  5. The method according to claim 4, wherein the antigen is the gag protein, the env protein or a part thereof of ATLV.
6. 該抗原が A T L Vの gag- env融合タ ンパク質である請求項 4記載 の方法。  6. The method according to claim 4, wherein the antigen is an ATLV gag-env fusion protein.
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WO2000020563A1 (en) * 1998-10-02 2000-04-13 Mitsubishi Chemical Corporation Method for inducing cellular immunity and cells with induced cellular immunity
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JP2004051582A (en) * 2002-07-23 2004-02-19 Ajinomoto General Foods Inc Immunostimulating composition containing mannose oligosaccharide
WO2006104199A1 (en) * 2005-03-29 2006-10-05 Tokai University Educational System Liposome composition for induction of immunity
US7294619B2 (en) 1994-08-29 2007-11-13 Wake Forest University Lipid analogs for inhibiting the activity of hepatitis B antigen
JP2008179563A (en) * 2007-01-24 2008-08-07 Cosmo Shokuhin Kk Functional material and functional food comprising useful phospholipid composition
WO2008105174A1 (en) 2007-02-28 2008-09-04 National University Corporation Hokkaido University Liposome for induction of cell-mediated immunity
JP2012232949A (en) * 2011-05-06 2012-11-29 Osaka Prefecture Univ pH-RESPONSIVE LIPOSOME
WO2014034046A1 (en) 2012-08-27 2014-03-06 国立大学法人帯広畜産大学 Vaccine formulation against toxoplasma gondii infection
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Cited By (14)

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US7294619B2 (en) 1994-08-29 2007-11-13 Wake Forest University Lipid analogs for inhibiting the activity of hepatitis B antigen
US5962437A (en) * 1994-08-29 1999-10-05 Wake Forest University Lipid analogs for treating viral infections
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US7294620B2 (en) 1994-08-29 2007-11-13 Wake Forest University Lipid analogs for inhibiting HIV-1 activity
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WO2000020563A1 (en) * 1998-10-02 2000-04-13 Mitsubishi Chemical Corporation Method for inducing cellular immunity and cells with induced cellular immunity
JP2004051582A (en) * 2002-07-23 2004-02-19 Ajinomoto General Foods Inc Immunostimulating composition containing mannose oligosaccharide
WO2006104199A1 (en) * 2005-03-29 2006-10-05 Tokai University Educational System Liposome composition for induction of immunity
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JP2008179563A (en) * 2007-01-24 2008-08-07 Cosmo Shokuhin Kk Functional material and functional food comprising useful phospholipid composition
WO2008105174A1 (en) 2007-02-28 2008-09-04 National University Corporation Hokkaido University Liposome for induction of cell-mediated immunity
US8821882B2 (en) 2008-09-17 2014-09-02 Obihiro University Of Agriculture And Veterinary Medicine Vaccine preparation for neospora caninum infection
JP2012232949A (en) * 2011-05-06 2012-11-29 Osaka Prefecture Univ pH-RESPONSIVE LIPOSOME
WO2014034046A1 (en) 2012-08-27 2014-03-06 国立大学法人帯広畜産大学 Vaccine formulation against toxoplasma gondii infection

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